Antibodies directed against receptor tyrosine kinases

Approximately 30 therapeutic monoclonal antibodies have already been approved for cancers and inflammatory diseases, and monoclonal antibodies continue to be one of the fastest growing classes of therapeutic molecules. Because aberrant signaling by receptor tyrosine kinases (RTKs) is a commonly observed factor in cancer, most of the subclasses of RTKs are being extensively studied as potential targets for treating malignancies. The first two RTKs that have been targeted by antibody therapy, with five currently marketed antibodies, are the growth factor receptors EGFR and HER2. However, due to systemic side effects, refractory patients and the development of drug resistance, these treatments are being challenged by emerging therapeutics. This review examines current monoclonal antibody therapies against RTKs. After an analysis of agents that have already been approved, we present an analysis of antibodies in clinical development that target RTKs. Finally, we highlight promising RTKs that are emerging as new oncological targets for antibody-based therapy.


Introduction
Antibodies represent an ideal approach for selectively interfering with a single target molecule, because of their high specificity. Therefore, therapeutic monoclonal antibodies (mAbs) have one of the most active and promising pipelines in the pharmaceutical industry, especially in the field of oncology. During the past 30 y, antibody cancer therapeutics have been developed and used clinically in an effort to realize the potential of targeted therapy. Therapeutic mAbs can lead to tumor cell death by different mechanisms of action. First, mAbs can block ligand-receptor growth and survival pathways by targeting antigens that are differentially expressed in tumor cells (tumor-associated antigens) and consequently can be used to block molecules involved in cancer progression or angiogenesis. Second, in cancer therapy, the innate immune effector mechanisms of mAbs lead to antibody-dependent cell-mediated cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP) of targeted cells. 1 Medical advances during the past 10 y have led to the introduction of 9 antibodies for the treatment of diverse cancers, with the antibodies targeting different tumor-associated antigens including surface glycoproteins associated with clusters of differentiation (CD), CTLA-A, or pathways regulated by growth factor receptors. 2 Five of these antibodies specifically and selectively target and block a receptor tyrosine kinase (RTK). RTKs are high-affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones that usually contain an intracellular kinase domain. This kinase function is the first element involved in the activation of one or several downstream intracellular signal transduction pathways. By targeting the extracellular part of the receptor protein kinase, the mAb is able to block the binding of the natural ligand, avoid conformational rearrangement essential to the activation of the kinase and thus the activation of the downstream signaling pathways, or activate immune effector functions. As key regulators of normal cellular processes and the development and progression of many types of cancer, RTKs are ideal cancer targets. In this review, we focus on non-conjugated mAb therapies developed against RTKs, with a brief analysis of agents that are already approved and used clinically, followed by a list of recent clinical and preclinical efforts toward new targets to develop novel immunotherapy approaches for cancers.

Currently Marketed Monoclonal Antibodies Targeting Receptor Tyrosine Kinases are Directed Against the Human Epidermal Growth Factor Receptor Family
Currently, the five antibodies on the market that target RTKs are directed against the human epidermal growth factor receptor family ( Table 1). The human epidermal growth factor receptor (HER) family members are structurally related and include Approximately 30 therapeutic monoclonal antibodies have already been approved for cancers and inflammatory diseases, and monoclonal antibodies continue to be one of the fastest growing classes of therapeutic molecules. Because aberrant signaling by receptor tyrosine kinases (rTKs) is a commonly observed factor in cancer, most of the subclasses of rTKs are being extensively studied as potential targets for treating malignancies. The first two rTKs that have been targeted by antibody therapy, with five currently marketed antibodies, are the growth factor receptors eGFr and Her2. However, due to systemic side effects, refractory patients and the development of drug resistance, these treatments are being challenged by emerging therapeutics. This review examines current monoclonal antibody therapies against rTKs. After an analysis of agents that have already been approved, we present an analysis of antibodies in clinical development that target rTKs. Finally, we highlight promising rTKs that are emerging as new oncological targets for antibody-based therapy.
EGFR (ERBB1), HER2/neu (ERBB2), HER3 (ERBB3), and HER4 (ERBB4), and all except HER3 contain an intracellular tyrosine kinase domain. All of the HER members other than HER2 are able to bind natural extracellular ligands. Seven ligands bind to EGFR including epidermal growth factor and transforming growth factor α; two ligands bind to HER3; and seven ligands bind to HER4. Activation of EGFR and HER2 (by its heterodimerization with HER3) induces a cascade of downstream signaling through several pathways, such as MAPK and PI3K/Akt/mTOR, resulting in cellular proliferation, differentiation, survival, motility, adhesion, and repair ( Fig. 1). 3 Mutations, overexpression or abnormal activation of receptors in the HER family are common features in several epithelial malignancies including lung, breast, stomach, colorectal, and head and neck cancers, pancreatic carcinoma and glioblastoma. 4 ANTI-HER2 Trastuzumab Trastuzumab (Herceptin®; Genentech, Inc.) was the first approved anti-RTK mAb. This approval has paved the way and proved the concept of targeting kinases with mAbs in cancer therapy. Trastuzumab is a humanized mAb that binds to the extracellular domain of the receptor tyrosine kinase HER2. 5,6 Trastuzumab was first approved in 1998 to treat early stage HER2-positive breast cancer, or metastatic breast cancer that substantially overexpresses HER2, and the approval was extended in 2010 to include HER2-positive metastatic cancer of the stomach or gastresophageal junction. The mechanisms underlying the anticancer activity of trastuzumab have not been completely elucidated. 7 However, several mechanisms have been proposed and there are generally accepted basic principles. First, trastuzumab does not block the dimerization of HER2 but its binding to its targeted receptor induces an inhibition of the intracellular signaling pathways (up to 50% over 5 d). 8,9 Second, trastuzumab downregulates the overall levels of HER2 on the cell surface. 10 Third, trastuzumab, by its binding to HER2, presents the cells to the immune system to allow ADCC of tumor cells, but the CDC process is not involved. 11,12 Through these three global mechanisms, treatment with trastuzumab leads to the inhibition of proliferation and the death of cells that overexpress HER2, induction of cell cycle arrest, and effects on cell adhesion, angiogenesis and the metastatic potential of tumor cells. [13][14][15] Pertuzumab With the understanding that HER3 is a necessary partner for HER2-mediated oncogenic activity in HER2-overexpressing tumors, the success and approval in 2012 of pertuzumab (Perjeta®/Omnitarg®; Genentech, Inc.), is not surprising. 16 Pertuzumab is a first-in-class HER2 dimerization inhibitor that acts by blocking ligand-induced HER2-to-HER3 heterodimerization and inhibiting HER3 signaling. 17,18 Pertuzumab is also able to inhibit heterodimerization of HER2 with the two other HER family members, EGFR and HER4, but preclinical observations have demonstrated that the blocking of HER2-HER3 likely represents the most relevant action of pertuzumab. 19,20 Other studies have also indicated that interfering with the HER3 component may be more relevant than inhibition of EGFR in HER2amplified breast cancer cell lines. 21 Similarly, high levels of HER3, rather than overexpression of HER2, were correlated with shorter overall survival in patients with ovarian cancer. 22 Pertuzumab is not a downregulator of the cell surface expression of HER2, but its binding to the tumor cells recruits the immune system and induces an ADCC process with only minor CDC effects. 23,24 Since the initial use of each anti-HER2 antibody alone as monotherapy or associated with chemotherapy, several studies have demonstrated the benefit of using trastuzumab and pertuzumab together. The co-localization of the two HER2 antibodies on distinct epitopes of the HER2 extracellular domain provides more efficient inhibition of tumor growth than one antibody alone. 23,25 ANTI-EGFR Cetuximab Given that EGFR is a transmembrane glycoprotein receptor that induces tumor cell proliferation and neovascularization, many studies have led to the development of either mAbs or small tyrosine kinase inhibitors as new agents targeting EGFR. 26,27 Studies have demonstrated that blocking EGFR signaling decreases growth and metastasis and also enhances the effect of chemotherapy. 28 Cetuximab (Erbitux®; Imclone Systems, Inc.) is a chimeric human-murine monoclonal IgG1 antibody that binds to the extracellular domain of EGFR and inhibits its dimerization. 29 The significant antitumor effect of cetuximab is mediated, in part, by inhibition of angiogenesis. 30,31 Cetuximab may elicit immunological responses such as ADCC or complement activation. [32][33][34] Several studies have demonstrated the in vivo anti-tumoral activity of cetuximab in diverse types of cancer and elucidated its mechanism of action. [35][36][37] For example, treatment with cetuximab influences cellular proliferation, apoptosis, and radiosensitivity in carcinomas of the head and neck. 38 Cetuximab is approved in many countries all over the world for treating patients with squamous cell carcinoma of the head and neck (SCCHN) and metastatic colorectal Cancer (mCRC). Continuous experimental and clinical investigations to better understand the activity of cetuximab have led to the discovery that a real benefit of treatment is obtained in patients with EGFR-expressing, RAS wild-type metastatic colorectal cancer. 39,40 The RAS mutation status is now used clinically to stratify patients likely to benefit from this therapy.

Panitumumab
In 2006, panitumumab (Vectibix®; Amgen, Inc.), a human antibody against EGFR, was approved by the US. Food and Figure 1. Overview of human epidermal growth factor receptor (Her) family signaling. Although the morphology of the extracellular domains of the four Hers are almost identical, their functional activity varies considerably. Her3 lacks inherent kinase function, but can heterodimerize with other Her receptors. Indeed, the Her2-Her3 dimer, which is considered to be the most active Her signaling dimer, is fundamental for Her2-mediated signaling in tumors with Her2 amplification. After ligand binding, the Her receptor becomes activated by dimerization between two identical receptors (i.e., homodimerization) or between different receptors of the same family (i.e., heterodimerization). Dimerization leads to the phosphorylation of several intracellular catalytic substrates, including members of the ras/raf/mitogen-activated protein kinase (MAPK) pathway, the phosphatidyl-3-kinase (PI3K)/Akt/PTeN family, and other important signaling pathways that regulate apoptosis, protein synthesis, and cellular proliferation. The monoclonal antibodies trastuzumab and pertuzumab are directed against Her2 members, with both leading to an inhibition of the Her2 signaling pathway but by two distinct mechanisms. Cetuximab and pertuzumab bind to the eGFr protein.
Drug Administration (FDA) for the treatment of patients with EGFR-expressing mCRC. By competing with endogenous EGF and TGFα ligand binding, panitumumab inhibits receptor phosphorylation and activation of cell signaling leading to blocking of EGF-dependent tumor growth mechanisms and also resulting in receptor internalization, apoptosis induction, inhibition of angiogenesis and autophagy. 41,42 In vivo, panitumumab not only blocks the formation of new tumors but also mediates therapeutic elimination of established tumors and acts cooperatively with chemotherapeutics in mediating tumor regression. 43 With an IgG2 isotype, panitumumab is not able to mediate ADCC or CDC. 44,45 Notably, panitumumab is not effective at recruiting NK cells and inducing NK cell-mediated ADCC but is effective at recruiting ADCC by myeloid effector cells (i.e., neutrophils and monocytes). 46,47 Based on the importance of RAS status for stratifying patients for treatment with cetuximab, several studies have demonstrated that the specific RAS mutation also determines the responsiveness to panitumumab treatment. 48,49 Further data are still needed to clarify the role of each specific RAS mutation for anti-EGFR-based treatments in mCRC and other types of cancers and this information should be considered for further clinical uses. 50 Nimotuzumab Nimotuzumab (h-R3), a humanized murine mAb, was developed at the Center of Molecular Immunology, Havana, Cuba. It has demonstrated antitumor activity similar to that of other anti-EGFR mAbs and shows promise as a single agent and as an adjunct to radiation. The mechanism of action of nimotuzumab involves not only direct targeting of EGFR in epithelial cancers and in vascular cells but also ADCC and CDC. 51 Surprisingly, the typical severe dermatological toxicities thus far associated with anti-EGFR therapy have not been described with nimotuzumab. 52 Experimental observations suggest that nimotuzumab requires bivalent binding leading to a more selective binding to cells that express moderate to high EGFR levels, rather than healthy cells expressing low levels of the RTK. 53 Nimotuzumab is presently approved in more than 20 countries for various indications including squamous cell carcinoma of the head and neck (SCCHN) in India, Cuba, Argentina, Colombia, Ivory Coast, Gabon, Ukraine, Peru, and Sri Lanka; for glioma (pediatric and adult) in Cuba, Argentina, Philippines, and Ukraine; and for nasopharyngeal cancer in China. Nimotuzumab has been designated as an "orphan drug" for the treatment of glioma by the FDA and for the treatment of glioma and pancreatic cancer by the European Medicines Agency (EMA). Currently, nimotuzumab is still being evaluated in Phase 3 clinical trials. 54,55

Continuous Efforts To Target Members of the HER Family: New Anti-HER Monoclonal Antibodies in Clinical Development
While trastuzumab, pertuzumab, cetuximab, panitumumab and nimotuzumab are still under investigation in clinical trials for different types of cancer, new agents directed against the same targets (EGFR and HER2) are entering clinical trials ( Table 2).
In parallel with such novel antibodies, next-generation versions of already approved products emerged. For example, CetuGEX TM (GT-MAB 5.2-GEX TM ) and TrasGEX TM (GT-Mab 7.3-GEX TM ) are antibodies produced by human glycoengineered cell lines (leukemic cells in suspension) that differ in their glycosylation capabilities and lead to the expression of biotherapeutics with human optimized glycosylation. CetuGEX TM and TrasGEX TM , glyco-optimized versions of cetuximab and trastuzumab, respectively, promised improved bioactivity and bioavailability and low/no immunogenicity. The increase in ADCC due to superior glycosylation in turn renders the improved antibodies suitable for treatment of patient sub-populations that were not responsive to the original product.
With the success of pertuzumab, which proved that targeting HER3 is a good way to treat cancer, some agents directed against HER3 are now being tested in clinical trials. 56 Today, HER4 remains the only member of the HER family for which no antibody is being developed clinically.

ANTI-EGFR
Still, the clinical interest in EGFR-targeting approaches is not decreasing, for two reasons: most, if not all, FDA approved EGFR-targeting agents have been associated with the development of chemoresistance in a consistent fraction of patients; and severe skin, renal, and gastrointestinal mucosa toxicities are commonly associated with the EGFR-targeting antibodies cetuximab and panitumumab. Furthermore, despite promising preclinical data, some anti-EGFR antibodies that entered in clinical trials failed to meet primary endpoints. In this way, the development of matuzumab (EMD72000), zalutumumab (HUMAX-EGFR), and imgatuzumab (RO5083945; GA-201; RG7160) was halted either in Phase 2 or Phase 3 because they did not demonstrate any benefit on predefined clinical endpoints of activity. For this reason, another challenge in discovering new EGFR targeted therapies is finding new biomarkers for efficient translation of preclinical data into clinical outcomes. Necitumumab (IMC-11F8/LY3012211) is a human IgG1 that has demonstrated similar antitumor potency to cetuximab, and had been presented as a safer therapeutic alternative. 57,58 Necitumumab is currently in Phase 3 clinical trials and the submission of the first marketing approval for necitumumab is expected before the end of 2014. Humanized mAb 806 (ABT-806) targets an EGFR deletion variant as well as a wild-type EGFR expressed in cells overexpressing the receptor. 59   has been shown to be superior to other anti-EGFR antibodies and tyrosine kinase inhibitors in treating tumors with specific deletion mutations or EGFR kinase domain mutations in vivo. As demonstrated in a Phase 1 first-in-human clinical trial, ABT-806 lacks normal tissue uptake or toxicity in cancer patients. 60 Sym004 is a mixture of two synergistic full-length anti-EGFR antibodies (futuximab and zatuximab), which bind to two separate non-overlapping epitopes on EGFR. 61 Both antibodies are involved in extensively cross-linking EGFRs on the cell surface leading to improved receptor elimination and induction of secondary effector functions. Sym004 has proved to be superior to available mAbs in several established animal models and has shown promising results in early human clinical trials. 62

ANTI-HER2
In contrast to the enthusiasm for the discovery of new and improved anti-EGFR therapeutic antibodies, there is less of a frenzy to develop new specific anti-HER2 antibodies. However, the limited efficacy of trastuzumab for cancer therapy, problems of acquired resistance and the potential effect of novel anti-HER2 antibodies as therapeutic partners or alternative therapeutics should justify more investment in HER2 targeting with novel mAbs. In addition to TrasGEX TM , the only other anti-HER2 mAb that has entered clinical trials is margetuximab (MGAH22). Margetuximab is a chimeric antibody with specificity and affinity similar to trastuzumab, with an Fc domain engineered to enhance ADCC. 63 ANTI-HER3 Antibodies directly targeting the HER3 extracellular domain are currently under clinical investigation. Seribantumab (MM-121/SAR256212), patritumab (U3-1287/AMG888) and LJM716 are human mAbs that target the HER3 receptor, inhibiting HER3 signaling and activation of associated survival pathways. [64][65][66][67] Other candidates that inhibit both ligand-dependent and ligand-independent HER3 signaling have been designed (AV-203, GSK2849330, REGN1400, KTN3379). 68 Therapeutic inhibitors of HER3 are currently being tested in several Phase 1 and Phase 2 clinical trials spanning various patient populations, and there is strong evidence that these agents have limited potential for single agent activity and should be considered as part of multidrug combinations. 69,70 In this context, RG7116/RO5479599 could be a highly potent HER3-targeting single agent because it was designed not only to block HER3 activation and induce HER3 downregulation but also to mediate enhanced ADCC by glycoengineering. 71

Clinically Investigated Monoclonal Antibodies Directed Against New Receptor Tyrosine Kinase Targets
New growth factor receptors with kinase domains, including c-MET, RON, IGF1R and EPHA2, EPHA3, are targeted by antibodies in cancer patients ( Table 2). 72,73 To prevent tumor growth, two pathways are also targeted by new mAbs: the formation of new microvessels, and more generally, the angiogenesis process is repressed by inhibiting VEGFR signaling; and stromal cells and the tumor microenvironment are altered using PDGFR, CSF1R and FGFR as targets.
Antibodies directed against the MET family ANTI-MET c-MET (mesenchymal-epithelial transition; HGFR [hepatocyte growth factor receptor]) is a membrane-spanning receptor tyrosine kinase. Upon binding with its only known ligand, hepatocyte growth factor (HGF), c-MET autophosphorylates and recruits several downstream effectors activating several biological processes including motility, proliferation, survival, invasion, and morphogenesis. 74,75 Consequently, the HGF/c-MET axis is implicated in a wide variety of epithelial, mesenchymal, and hematological malignancies, rendering c-MET an attractive target for cancer therapies. 74,76 There are currently several HGF/c-MET inhibitors under clinical evaluation, with four antibodies directed against the extracellular part of c-MET. Onartuzumab (MetMab/OA-5D5) is a one-armed monovalent humanized antibody designed to prevent receptor oligomerization and initiation of signaling frequently observed with antibodies against c-MET. Onartuzumab reduces c-MET phosphorylation and diminishes c-MET density on the cell surface and is currently the most advanced of the anti-MET antibodies, with Phase 3 clinical trials in progress. 77,78 However, a Phase 3 study (METLung study), evaluating onartuzumab in combination with erlotinib (Tarceva®; Roche), a small chemical inhibitor of EGFR tyrosine kinase activity, compared with erlotinib alone has recently been stopped due to a lack of clinically meaningful efficacy of ornatuzumab. 79 Even if some data from this study have yet to be extensively analyzed and reviewed, these disappointing findings challenge the future development of onartuzumab and revive the race to find clinical benefits of using anti-MET antibodies. Although almost all major human cancers seem to harbor some dysregulation of the MET pathway, only a minority of tumors are 'addicted to MET' mainly because tumor cells develop many concomitant or staggered resistance pathways. 80 Greater benefits would probably be observed if clinical trials are designed with prospective evaluation or better targeting of the specific subpopulation of drug-resistant patients, or by staggering treatment. Other humanized mAbs with high affinity for c-MET are ABT-700 (h224G11) and LY2875358 (LA480), which inhibit the major functions of c-MET, including not only cell proliferation, migration, and invasion, but also cell scattering and angiogenesis. 81,82 By inducing c-MET downregulation and triggering ADCC functions, both are promising candidates for the treatment of ligand-dependent and ligand-independent tumors as single or combined therapies. 83 ARGX-111 is a human mAb that potently neutralizes both ligand-dependent and independent receptor signaling of c-MET with enhanced ADCC by Fc engineering.
ANTI-RON Given the increasing interest in developing MET inhibitors to fight cancer, it is not surprising that new therapeutics are now being developed to target RON, another member of the MET family. 84 RON (recepteur d'origine nantais) or MS1R (macrophage stimulating 1 receptor) shares many common structural and biochemical features with MET. Ligand-dependent or independent activation of RON results in cell proliferation, migration, and matrix invasion, collectively known as invasive growth, as occurs with activation of c-MET. 85 Intensive study has led to the preclinical evaluation of several anti-RON antibodies, with narnatumab (IMC-RON8) being the first to enter clinical trials targeting RON overexpression. Narnatumab is a human mAb that prevents binding of RON ligand, downregulates RON expression, hinders ligand-induced cell migration and reduces cell transformation. 86 Antibodies directed against the Insulin receptor family Similar to the EGFR pathway, the insulin-like growth factor receptor (IGFR) signaling system is comprised of multiple circulating ligands, such as IGF-I, IGF-II, and insulin, which interact with membrane-bound receptors, such as type I IGF receptor (IGF1R) and insulin receptor (IR). 87 Most anti-IGFR pathway agents in clinical development are targeted against the IGF1R, which contains an intracellular tyrosine kinase domain capable of recruiting insulin-receptor substrates (IRS) or Src homology 2 domain-containing (Shc) proteins. IGF1R activation has been identified as playing a major role in the development, maintenance, and progression of cancer. 88,89 There are several IGF1R-targeting agents undergoing clinical testing; all of these agents have a similar mechanism of action, with binding to IGF1R that causes receptor internalization, and thereby prevents binding of ligand to receptor by removing receptors from the cell surface. 90 Such anti-IGF1R mAbs have demonstrated broad preclinical antitumor activities. 91,92 However, their effectiveness in clinical trials has been limited and none have been approved yet by the FDA for general oncological use. 93 In the past, several agents failed to demonstrate a statistically significant improvement in the primary end point in clinical trials. For example, figitumumab (CP-751871) and teprotomumab (R1507) failed in Phase 3 for NSCLC; ganitumumab (AMG-479) failed to show a benefit in pancreatic adenocarcinoma patients; cixutumumab (IMC-A12/LY3012217) presented mixed results in Phase 2 and has not progressed to Phase 3; robatumumab (Sch717454) in advanced NSCLC or AVE1642 in multiple myeloma have demonstrated insufficient activity to deserve further development in this subgroup of patients. The development of BIIB022, in contrast, was terminated due to a non scientific decision of the sponsor company. Some of these antibodies, such as ganitumab and cixutumumab, are still being evaluated in Phase 1 and Phase 2 trials for oncological indications. Clinical trials (Phase 2) are ongoing with a fourth anti-IGF1R mAb. Dalotuzumab (MK-0646; h7C10), a recombinant humanized IgG1 mAb, acts by inhibiting IGF1R ligand-mediated tumor cell proliferation, and has proven preclinical efficacy in multiple cancer cell lines and in mouse xenograft models. 94 Antibodies directed against the Ephrin receptor family The 16 members of the Ephrin receptor superfamily of RTKs are key players in various signaling pathways and mediate critical steps in a wide variety of physiological and pathological processes. The members of the Ephrin Receptors superfamily are divided into two groups, EPHA and EPHB, based on the types of ligands (ephrins) they bind. EPH receptors are associated with tumor growth, invasiveness and metastasis. 95 The function of an EPH receptor in oncogenesis can be a consequence of either its upregulation or overexpression or its downregulation. 96 Despite the considerable complexity of EPH signaling, EPH receptors have emerged as potential therapeutic targets. 97,98 The development of several antibodies targeting EPHA2 (agonist), EPHB2 (antagonist) or EPHB4 (antagonist) was prematurely stopped due to a lack of antitumor activity or a limited therapeutic window in preclinical models. Only two antibodies targeting an EPH receptor are currently in clinical trials (Phase 1): an anti-EPHA2 (DS-8895a) and an anti-EPHA3 (KB004/IIIA4). Little is known about DS-8895a because no public data are available. In contrast, published data has described encouraging preclinical results, mainly in hematological malignancies, for KB004, a nonfucosylated human IgG1 derived from the agonistic anti-EPHA3 IIIA4 antibody. 73 Antibodies directed against the VEGFR family The first observation, by Folkman in the 1970s, that tumors are unable to grow beyond 2 mm 3 unless they are supported by neovascularization, resulted in intensive study to understand the angiogenesis mechanism in tumor growth. In this context, vascular endothelial growth factors and receptors (VEGF, VEGFR) emerged as principal angiogenesis actors and have been validated as targets in cancer therapy. 99 The VEGF family is comprised of the ligands placental growth factor (PlGF), VEGF-A, VEGF-B, VEGF-C, and VEGF-D, and the receptors VEGFR1, VEGFR2, and VEGFR3. Several anti-VEGFR mAbs are now in clinical development ( Table 2).

ANTI-VEGFR1
Although VEGF-A is known to bind and activate both VEGFR1 and VEGFR2, VEGFR1 is the only known receptor for the ligands PlGF and VEGF-B. In human cancer, VEGFR1 mediated signaling is responsible for several cancer pathogenesis mechanisms including direct tumor activation, activation of nonmalignant supporting cells and angiogenesis. 100 Icrucumab (IMC-18F1/LY3012212) is a human IgG1 mAb that was designed to target VEGFR1. This antibody potently inhibits ligand-dependent phosphorylation of VEGFR1 by blocking the binding of VEGF-A, VEGF-B and PlGF and downstream signaling. 101 ANTI-VEGFR2 VEGF-A interaction with VEGFR2 has been demonstrated to be an essential modulator of tumor angiogenesis based on the results of an extensive range of human and preclinical investigations. Ramucirumab (IMC-1121B/ LY3009806) is a human IgG1 specific for the VEGFR2 receptor. By antagonizing endogenous VEGF signaling, ramucirumab inhibits the most prominent interactions between neoplastic and stromal cells, i.e., neo-angiogenesis. 102,103 A decision from the FDA on ramucirumab as a second-line single-agent treatment option for patients with advanced gastric cancer is anticipated in the spring of 2014. Ramucirumab has also been explored in Phase 3 trials as a treatment for colorectal cancer, hepatocellular carcinoma, and lung cancer and is currently under evaluation for other oncological indications. 104,105 Tanibirumab (TTAC-0001) is a human mAb that specifically binds to VEGFR2 and prevents the binding of its ligand. Tanibirumab has demonstrated antitumor activity against various cancer models, including lung, breast, colorectal and glioblastoma models, and is being studied in Phase 1 clinical trials. 106

ANTI-VEGFR3
VEGFR3 and its ligands, VEGF-C and VEGF-D, control tumor lymphangiogenesis by enhancing proliferation and survival of lymphatic endothelial cells. VEGFR3 levels are upregulated in tumor vessels, and inhibitors blocking VEGFR3 homodimerization, VEGFR3/VEGFR2 heterodimerization, or VEGF-C binding inhibit tumor angiogenesis in culture and in mice. 107 Based on these observations, IMC-3C5 (hF4-3C5), a human IgG1 mAb, was designed to bind VEGFR3 and block the binding of VEGF-C and VEGF-D ligands to the receptor, thereby inhibiting subsequent signaling. 108 However, with results from the literature suggesting that a dual blockade of both VEGFR2 and VEGFR3 simultaneously may represent a more potent approach to effective cancer therapy than inhibiting VEGFR3 alone, the clinical efficacy of IMC-3C5 should probably be investigated in association with an anti-VEGFR2 or an anti-VEGF therapy. 108,109 Antibodies directed against the KIT family The KIT family of receptor tyrosine kinases includes c-Kit, Flt3 and fms/CSF1R. The c-Kit receptor plays a key role in tumor occurrence, development, migration and recurrence. 110 Although targeted drugs have emerged to inhibit the kinase activity of this receptor, biotherapeutics are mainly directed against CSF1R. Also known as macrophage colony-stimulating factor receptor (M-CSFR), CSF1R is a cell-surface receptor for its ligands, colony-stimulating factor 1 (CSF1) and Interleukin-34 (IL-34). CSF1R plays a vital role in monocyte-macrophage generation, survival, and function and as a regulator of tumorassociated macrophages (TAMs) and tumoral cell growth. Two anti-CSF1R antibodies are being investigated in Phase 1 clinical trials. IMC-CS4 was shown to trigger in vitro depletion of TAMs, and RG7155 (RO5509554) was demonstrated to enhance the efficacy of chemotherapy.

Antibodies directed against the PDGFR family
The PDGF family consists of five isoforms that are homodimers of A-, B-, C-, and D-polypeptide chains, and a heterodimer AB. The five PDGF ligands act via binding to PDGFRα and PDGFRβ. PDGFRα and PDGFRβ associate as homo-or heterodimers, each inducing similar, but not identical, cellular effects. PDGFR has attracted increasing attention as a potential target of anti-tumor therapy for several types of cancer. 111 PDGFRα is expressed frequently by tumor-associated stromal cells and by cancer cells in a subset of tumors. Olaratumab, IMC-3G3, a human IgG1 mAb, specifically binds to the human PDGFRα with high affinity, which blocks ligand binding and receptor activation. 112 Olaratumab has already been evaluated in several types of cancer in preclinical studies and is currently being evaluated in Phase 2 clinical trials for patients with several types of solid malignancies. 113 After a Phase 2 stage on glioblastoma multiforme and NSCLC, the development of tovetumab (MEDI-575), a humanized IgG2 mAb directed against the PDGFRα, was stopped with undisclosed reasons.

Antibodies directed against the FGFR family
The fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling pathway plays a fundamental role in many physiological processes by orchestrating angiogenesis. The FGFR cascades play crucial roles in tumor cell proliferation, angiogenesis, migration, and survival, thereby providing a strong rationale for the development of anti-FGF/FGFR agents. 114,115 Until the discovery of the different mutation and expression status of each of the FGFR isoforms in cancer, a plethora of small molecule kinase inhibitors, either selective or pan-FGFR (FGFR1-4), are being investigated in clinical trials. 116,117 Although anti-FGFR mAbs have been assessed and their efficacy has been demonstrated in preclinical cancer models, little information is available on the tolerability profile of any of these agents. The first FGFR antibody to enter clinical development was the anti-FGFR3 antibody RG7444/MFGR1877S (but its development seems to be paused because the Phase 1 clinical trial was terminated) followed by the anti-FGFR2 antibody BAY 1179470.

Future Emerging Promising Targets in the Preclinical Stage
Because aberrant signaling by RTKs is an etiologic factor in cancer, most (if not almost all) of the other subclasses of RTKs are being studied as potential targets for treating malignancies. For example, the angiopoietin/Tie ligand/receptor system (especially angiopoietin2/Tie-2) emerged as an anti-angiogenic alternative approach to anti-VEGF pathway therapies. 118,119 Receptor tyrosine kinase-like orphan receptor (ROR) targeting is anticipated as a safe approach to selectively attack a tumor much more forcefully than healthy tissue. 120,121 Anaplastic lymphoma kinase (ALK)-targeted immunotherapy was proposed as a promising therapeutic strategy for cancer therapy. 122,123 Currently, the most promising anti-cancer targets appear to be the members of the TAM receptor tyrosine kinase family (Axl, Tyro3 and Mer). 124,125 Even if Tyro-3 is an interesting target, in this family, Axl and Mer are particularly under the spotlight, 126 and many studies have demonstrated their crucial role in cancer as major players in the tumor microenvironment. [127][128][129][130] Moreover, both are involved in the drug-resistance process and their inhibition clearly enhances sensitivity of cancer cells to cytotoxic agents. [131][132][133][134] In this context, joint efforts have been made to preclinically validate the efficiency of anti-Axl antibodies. [135][136][137] Potent therapeutic mAbs targeting Axl are currently in the discovery and preclinical stages of development and demonstrate highly promising results (unpublished in-house data).

Emerging New Approaches to Enhance Treatment Efficacy
Given that most of patients develop resistance within one year after initiation of anti-HER2 or anti-EGFR treatment, there is still a great need for alternative treatments to overcome this acquired resistance. 138,139 The molecular mechanisms by which tumor cells may adapt and escape after HER2 or EGFR inhibition are similar: appearance of mutations, upregulation of downstream signaling pathways or activation of alternative pathways. 80,140 Extensive crosstalk among receptor kinases suggests that blocking signaling from more than one family member or from different families may be essential to effectively treating cancer and limiting drug resistance. Strategies that should prevent and overcome resistance include targeting several RTKs with the same antibody (bispecific antibodies), targeting the same RTK with different antibodies, or combined targeting of two or more different RTKs.

Bispecific antibodies
The clinical efficacy of bispecific antibodies is currently being investigated ( Table 3). Among these promising types of antibodies, we can cite the dual EGFR/HER3 duligotumab (MEHD7945A) that presents a greater antitumor potency than cetuximab and retained potent activity in tumors refractory to EGFR inhibitors; the dual HER2/HER3 MM-111 antibody that could prevent the development of HER3-mediated resistance to currently existing anti-HER2 therapies; and the dual IGF1R/ HER3 MM-141 antibody that displays enhanced in vitro and in vivo activity relative to inhibitors of either pathway in isolation, and synergizes with targeted and chemotherapies. [141][142][143] Ertumaxomab is a trifunctional antibody (trAb) that bivalently targets HER2 and the T cell specific CD3 antigen, and its Fc region is selectively recognized by immune cells. Ertumaxomab is a promising novel anticancer biologic that links innate with adaptive immunity. 144,145 Treatment with ertumaxomab might complement the therapeutic activity of chemotherapy and other anti-HER2/anti-EGFR receptor reagents. 146,147 Antibody combination Preclinical data have shown that the use of two or more inhibitors against the same EGFR family member leads to synergistic anti-tumor activity. 148,149 This dual-targeting approach has been successfully translated to the clinic with the combination of trastuzumab, pertuzumab and docetaxel for HER2-positive breast cancer. The Sym004 or MM-151 mixtures, previously described in this review, are the lead examples of this category of therapeutics. The synergistic anti-tumoral effect observed by associating antibodies able to target non-overlapping epitopes of a defined RTK is most probably due to their ability to increase the RTK downregulation. 150 Although the receptor cell surface expression is reduced, combination therapy seems to be able to further improve the ADCC process as demonstrated in the trastuzumab and pertuzumab combination. 151 This approach is currently being evaluated on other RTK family members, including the combination of two antibodies directed against IGF1R. 152 Even if this strategy will increase efficacy and overcome acquired resistance due directly to the targeted RTK pathway, it may lack efficacy when the resistance appears by indirect activation of an alternative pathway. Consequently, one of the most promising strategies to fight resistance is to combine antibodies targeting distinct RTK families. IGF1R, MET and AXL pathways are described as the main alternative RTK pathways leading the tumor to become resistant. 140 Given that, we could expect higher activity and less resistance combining current anti-EGFR or anti-HER2 therapies with anti-MET, anti-IGF1R or anti-AXL antibodies. With this aim in mind, ganitumab used in combination with panitumumab is currently under evaluation in a Phase 1 clinical trial.

Antibody-drug conjugates
The majority of unconjugated mAbs are used in combination with conventional chemotherapy drugs and radiotherapy. As an example, it is well known that cetuximab enhances the sensitivity  of tumor cells to chemotherapy and to radiation. However, this concurrent administration results in interplay and exacerbation between the adverses effects of the individual agents. 153 Therefore, significant effort has been devoted to improving mAbs through various modifications to achieve the efficacy of combination treatments while minimizing adverse effects. One idea was to conjugate mAbs either with a radioactive substance (radioimmunotherapy), drug (chemolabeled mAbs), or a cytotoxin (immunotoxins) to generate conjugated mAbs or antibodydrug conjugates (ADCs) capable of antigen-specific delivery of highly potent cytotoxic molecules to tumor cells. ADCs are a niche class of drugs that offer promise, particularly as oncology drugs. 154 Several ADCs are already in clinical development for cancer treatment. Ado-trastuzumab-emtansine (Kadcyla®), a chemolabeled antibody, is the first approved ADC targeting a receptor tyrosine kinase (approved in 2013 by the FDA and the EMA). Ado-trastuzumab-emtansine is indicated for the treatment of patients with refractory HER2-positive locally advanced or metastatic breast cancer. Given all of the advantages previously described of targeting RTKs in oncology and the high clinical value of ado-trastuzumab-emtansine, it is not surprising that other ADCs in this domain are being developed ( Table 4). Until now, the clinical success of immuno-conjugates in oncology has been very limited compared with naked mAbs. However, with the evolution of the research on ADCs, we speculate that the number of ADCs entering clinical trials will rapidly accelerate in the coming years.

Conclusion
In the last decade, due to intensive innovative discovery of novel cancer therapies, high-profile approved agents entered the market. Advances in molecular biology have led to an explosion in the number of potential cancer targets and a rich pipeline of anticancer drugs. However, cancer is an extremely complex set of diseases, both anatomically and molecularly, and with only 15-20% of clinical candidates reaching the market, cancer continues to be an area of high unmet medical need, with many potential cancer targets still lacking drugs and drug resistance remaining as an enduring problem.
Despite the recognized success of the five clinical antibodies currently directed against the receptor tyrosine kinases EGFR and HER2, there are still many remaining issues. Overcoming systemic side effects and the generation of resistance would be a significant improvement for the pharmaceutical industry and for patients. Thus, in addition to new anti-HER antibodies in development, new RTKs involved in tumor progression, angiogenesis or stromal cells are emerging as oncological targets for antibody therapies.
In this review, we decided to focus on unconjugated antibodies that directly target RTKs. However, with the success of bevacizumab (an anti-VEGF antibody developed by Genentech and marketed under the name Avastin®), inhibition of some of these RTKS can also be indirectly accomplished by ligand trapping (for example, antibodies are being developed against HGF, FGF, IGF, and others). 155,156 Moreover, although the efficacy of unconjugated antibodies has been demonstrated, molecular genetics and chemical modifications to mAbs have advanced their clinical utility. Derivatives of mAbs, produced by modification of immune effector engagement or by conjugating cytotoxic agents to mAbs, have improved pharmacokinetic profiles and enhanced targeted therapeutic delivery to tumors. The increasing abilities of antibody modifications will continue to make it possible to construct more diverse and efficacious mAb-based therapeutics.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.